WO2016140528A1 - Immunocytokine conjuguée à un variant d'interféron bêta humain et son procédé de préparation - Google Patents

Immunocytokine conjuguée à un variant d'interféron bêta humain et son procédé de préparation Download PDF

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Publication number
WO2016140528A1
WO2016140528A1 PCT/KR2016/002129 KR2016002129W WO2016140528A1 WO 2016140528 A1 WO2016140528 A1 WO 2016140528A1 KR 2016002129 W KR2016002129 W KR 2016002129W WO 2016140528 A1 WO2016140528 A1 WO 2016140528A1
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Prior art keywords
beta
immunocytokine
human interferon
antibody
interferon
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PCT/KR2016/002129
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English (en)
Korean (ko)
Inventor
신영기
김영덕
최준영
김태은
이용진
이주호
김영문
Original Assignee
에이비온 주식회사
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Application filed by 에이비온 주식회사 filed Critical 에이비온 주식회사
Priority to JP2017546891A priority Critical patent/JP6779896B2/ja
Priority to ES16759164T priority patent/ES2839404T3/es
Priority to CN201680015754.1A priority patent/CN107438624B/zh
Priority to EP16759164.3A priority patent/EP3266801B1/fr
Publication of WO2016140528A1 publication Critical patent/WO2016140528A1/fr
Priority to US15/693,148 priority patent/US10806799B2/en
Priority to US17/023,207 priority patent/US20210009720A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/565—IFN-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/215—IFN-beta
    • A—HUMAN NECESSITIES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6845—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6875—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the present invention relates to an immunocytokine conjugated with a human interferon-beta mutant and a method for preparing the same, and more specifically, an immunocytoprotein in which an interferon-beta mutant and an antibody are combined with superior activity and function than a native interferon-beta.
  • the present invention relates to a caine and a method for producing the same.
  • Mab as a diagnostic and therapeutic reagent for various disease states.
  • Recombinant DNA techniques have been used to prepare chimeric or humanized antibodies for administration to humans.
  • monoclonal antibodies are marketed and used, for example, as RITUXAN®, HERCEPTIN®, AVASTIN®, etc. for the treatment of cancer, infectious diseases, immune diseases, and the like.
  • Monoclonal antibodies are targeted molecules and can be localized within specific crop regions (cells, tissues, etc.), such as pathological tissue. Due to this property, it is specific at the pathological tissue site. Efforts to target molecules have also resulted in the development of Mabs conjugated to various substances (payloads).
  • Such materials can be roxine, drugs, radionuclides, prodrug compounds, and the like. Many of these bindings involve chemical conjugation of reactive moieties (payloads) with specific preparations of antibodies, processes that can be cumbersome and prone to modification (US 4,671,958).
  • immunocytokines are of particular interest.
  • the cotton histokinin means a fusion protein comprising an antibody and a cytokine. Such proteins possess both antigen-binding capacity and cytokine activity.
  • Cytokines are a category of signaling proteins and glycoproteins that are widely used for intercellular communication, such as hormones and neurotransmitters.
  • cytokines While hormones are secreted into the blood by specific organs and neurotransmitters are involved in neuronal activity, cytokines are a more diverse group of compounds in terms of origin and purpose. It is produced by a wide range of hematopoietic and non-hematopoietic cell types, can affect nearby cells or affect the entire organism, and is sometimes strongly dependent on the presence of other chemicals.
  • the cytokine family consists primarily of smaller water soluble proteins and glycoproteins with a mass of 8 to 30 kDa. Cytokines are important for the functionalization of both innate and degenerative immune responses. Often cytokines are secreted by immune cells that have met the pathogen, activating and recruiting more immune cells and increasing the system response to the pathogen.
  • interferon In cytokineosis, interferon (IFNs) is a type of cytokine that has antiviral activity, inhibits cell proliferation, and modulates the natural immune response
  • interferon-beta IFN- ⁇
  • IFN- ⁇ interferon-beta
  • Mult iple sclerosis Treatment opt ions for pat ients wi th re 1 aps i ng-r em i 11 i ng and secondary progressive mult iple sclerosis, 1999).
  • interferon-beta has antiviral activity, cell growth inhibition or anti growth activity, lymphocyte cytotoxicity activity, immunomodulatory activity, target cell differentiation or inhibition activity, macrophage activation activity, and increased cytokine production.
  • the activity may be increased when sugar chains are added.
  • human interferon-beta variants having increased activity or function by introducing sugar chains into human natural interferon-beta, a glycoprotein, have been disclosed in Korean Patent Publication No. 10-0781666. Therefore, in order to use the human interferon-beta mutant which shows the efficacy superior to the pharmacological effect of the natural type interferon-beta, it is necessary to develop the immunocytokine conjugated with the antibody and to obtain it in high yield. Is required.
  • the inventors of the present invention have invented immune cytokines in which human interferon-beta variants with increased or enhanced activity or function by introducing sugar chains into human native interferon-beta, and antibody-binding host cells of such immunocytokines. It was found that the expression level in E. coli was significantly improved compared to the immunocytokine conjugated with the native type interferon-beta and the antibody, thereby completing the present invention.
  • an object of the present invention to provide a method for the preparation of (a) human interferon-beta variants; And (b) the phosphorus An immunocytokine comprising an antibody or fragment thereof covalently linked to a hepatic interferon-beta variant directly or indirectly, wherein the human interferon-beta variant comprises (i) a polypeptide comprising the entire amino acid sequence set forth in SEQ ID NO: 1; ii) a polypeptide comprising a substantial portion of the amino acid sequence set forth in SEQ ID NO: 1, (iii) a polypeptide selected from the group consisting of polypeptides substantially similar to the polypeptides of (a) or (b) and having human interferon-beta activity
  • an immunocytokine which is a polypeptide comprising an N-linked sugar chain.
  • Another object of the present invention is to provide a polynucleotide encoding the immunocytokine.
  • Another object of the invention is (a) a human interferon-beta variant; cloning a polynucleotide encoding a fusion polypeptide comprising (b) a peptide linker and (c) an antibody or fragment thereof into an expression vector;
  • the human interferon-beta variant is to provide a method for increasing the production of target specific human interferon-beta, characterized in that consisting of a peptide sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 4.
  • Another object of the present invention is to provide a vector comprising the polynucleotide.
  • Another object of the present invention is to provide a host cell transformed with the vector.
  • Another object of the present invention is to prepare an immunocytokine by (a) providing a host cell, (b) culturing a provided cell, and (c) recovering an immunocytokineol from the cell or culture medium. It provides an immunocytokine manufacturing method comprising.
  • the present invention to achieve the above object is (a) a human interferon-beta variant; And (b) an immunocytokine comprising an antibody or fragment thereof covalently linked directly or indirectly to the human interferon-beta variant, wherein the human interferon-beta variant comprises (i) the entire amino acid sequence set forth in SEQ ID NO: 1; A polypeptide comprising (i) a polypeptide comprising a substantial portion of the amino acid sequence set forth in SEQ ID NO: 1, (iii) a human interferon-beta selected from the group consisting of a polypeptide substantially similar to the polypeptide of (a) or (b) above As an active polypeptide, an immunocytokine which is a polypeptide comprising an N-linked sugar chain is provided.
  • a human interferon-beta variant cloning a polynucleotide encoding a fusion polypeptide comprising (b) a peptide linker and (c) an antibody or fragment thereof;
  • the human interferon-beta variant is to provide a method for increasing the production of target specific human interferon-beta, characterized in that consisting of a peptide sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 4.
  • the present invention is to provide a polynucleotide encoding the immunocytokine.
  • the present invention is to provide a vector comprising the polynucleotide.
  • the present invention is to provide a host cell transformed with the vector.
  • the present invention provides a (a) providing a host cell It provides a method for producing an immune cytokine comprising the steps of (b) culturing the provided cells and (c) recovering the immunocytokines from the cells or culture medium.
  • a method for producing an immune cytokine comprising the steps of (b) culturing the provided cells and (c) recovering the immunocytokines from the cells or culture medium.
  • the present invention will be described in detail.
  • the therapeutic potential of cytokines is often limited by severe side effects that occur at low concentrations, thereby preventing the presence of striking cytokine concentrations in target tissues. Accordingly, it is possible by immunocytokines to target cytokines and deliver them to disease sites using antibodies to increase the therapeutic potential of cytokines and protect normal tissues from toxic effects.
  • the immunocytokine according to the present invention is a variant of human interferon-beta, and is a cytokine whose activity or function is increased or improved by introducing a sugar chain into a native interferon-beta.
  • the inventors of the present invention if the human interferon-beta variant is combined with an antibody and can be used for the target therapy of multiple sclerosis, viral diseases, etc. in the form of an immunocytokine, the natural interferon-beta 7 ⁇ conjugated immunocytokine and Compared to the present invention, the present invention has been completed.
  • the present invention provides a kit comprising (a) human interferon-beta variants; And (b) an immunocytokine comprising an antibody or fragment thereof covalently linked directly or indirectly to the human interferon-beta variant, wherein the human interferon-beta variant comprises (i) the entire amino acid sequence set forth in SEQ ID NO: 1; A polypeptide comprising (i) a polypeptide comprising a substantial portion of the amino acid sequence set forth in SEQ ID NO: 1; and (iii) a human interface selected from the group consisting of a polypeptide substantially similar to the polypeptide of ( a ) or (b) above.
  • an immunocytokine which is a polypeptide comprising an N-linked sugar chain.
  • the human interferon-beta variant increased or improved in activity or function as compared to the native human interferon-beta, wherein the glycine-asparagine at the C-terminus of its amino acid sequence in the native human interferon-beta or the native human interferon-beta variant.
  • An isoleucine-threonine-valine sequence, N-linked to an asparagine residue of said additional sequence It is characterized in that it comprises a type sugar chain.
  • the "natural human interferon-beta variant” includes all polypeptides having all or a portion of amino acid sequences derived from native human interferon-beta and having human interferon-beta activity. That means it.
  • the term "activity of human interferon-beta” as defined above is defined as one or more activities that are sufficient to identify any polypeptide as human interferon-beta among those known to have human interferon-beta. Such activities may include alleviation, alleviation or therapeutic activity against multiple sclerosis, antiviral activity, cell growth inhibitory activity, anti-growth activity, anti-proliferative activity, lymphocyte cytotoxicity activity, immunomodulatory activity, target cell as described above.
  • the "polypeptide having all or part of the amino acid sequence derived from natural human interferon-beta” includes the whole or substantially part of the amino acid sequence of SEQ ID NO: 1 which is the amino acid sequence of the natural human interferon-beta.
  • polypeptide or a polypeptide substantially similar to such a polypeptide wherein the "polypeptide comprising a substantial portion of the entire amino acid sequence of SEQ ID NO: 1" means a native human interferon having the amino acid sequence of SEQ ID NO: 1.
  • -A polypeptide comprising a portion of the amino acid sequence of SEQ ID NO: 1, which has a dong or more activity compared to -beta, or which has a low activity but still retains the activity of human interferon-beta
  • SEQ ID NO: 1 All or a substantial portion of the amino acid sequence disclosed in Polypeptide substantially similar to 'human' having the same or more activity or lower activity than the native human interferon-beta of SEQ ID NO: 1 It is defined as a polypeptide comprising all or a substantial portion of the amino acid sequence of SEQ ID NO: 1, which contains one or more substituted amino acids while still retaining the activity of interferon-beta.
  • the N-terminal portion, the C-terminal portion, or a substituted amino acid is involved in a motif essential for the activity of human interferon-beta, thereby depleting the N-terminal portion and / or C-terminal portion.
  • a polypeptide comprising a substituted amino acid does not exhibit the activity of human interferon-beta, but nevertheless, it is confirmed that the above polypeptide derived from SEQ ID NO: 1 has one or more of the above-exemplified activities.
  • / or other methods related to human interferon-beta identification known in the art based on the filing of the present invention it is possible to distinguish and detect such inactive polypeptides from the active polypeptides.
  • the human interferon-beta variant of the present invention comprises a glycine-aspara long-isoleucine-threonine-valine sequence at the C-terminus and an N-linked sugar chain at the position thereof, wherein the human interferon-beta active
  • the following arginine (R27) site of the polypeptide or wild-type interferon beta may be defined as either one which is mutated to threonine (R27T) or to serine (R27S), more preferably SEQ ID NO: 2 to sequence It means a polypeptide comprising the amino acid sequence of any one of No. 4.
  • the human interferon-beta variant of the present invention comprises a glycine-asparagine-isorucin-threonine-valine sequence at the C-terminus thereof. It is to be understood as all polypeptides having human interferon-beta activity, including N-linked sugar chains in position.
  • the human interferon-beta variant of the present invention includes all glycine-aspara long-isoleucine-threonine-valine sequences at the C-terminus and all human human interferon-beta activities having an N-linked sugar chain therein. Defined as a polypeptide. More preferably, the 'human interferon-beta variant' of the present invention may be a mutein of interferon-beta having an amino acid sequence of any one of SEQ ID NOs: 2 to 4, and named by the present inventors carbiferon. It became. Carbiferon of the present invention takes the form of one to two sugar chains added to the natural interferon-beta.
  • arginine (R), which is amino acid 27, is replaced with threonine (T) or serine (S), or the native human interferon-beta
  • T threonine
  • S serine
  • a C-terminus is meant a polypeptide comprising a glycine-asparagine-isoleucine-threonine-valine (GNI-TV) sequence and having an N-linked sugar chain therein.
  • Human interferon-beta variants show enhanced or increased antiviral activity, cell growth inhibitory activity, immunomodulatory function, and half-life in vivo compared to native interferon-beta.
  • SEQ ID NO: 2 is an amino acid sequence of R27T which is an interferon ⁇ variant
  • SEQ ID NO: 3 is an interferon ⁇ variant R27S in which amino acid No. 27 of SEQ ID NO: 1 is substituted with S
  • SEQ ID NO: 4 is the amino acid sequence of the interferon ⁇ variant GNITV including the GNITV amino acid after the stop codon.
  • the SEQ ID NO: 1 to 4 includes the start codon at the ⁇ terminal, the protein of SEQ ID NO: 1 to 4 can be omitted when the linking with the other linker (linking the C terminal of the linker and the ⁇ terminal of the carbiferon).
  • Antibodies in the present invention are broadly used and include monoclonal antibodies, polyclonal antibodies, polyspecific antibodies (eg, bispecific antibodies), and antibody fragments (as long as they exhibit the desired antigen-binding activity). It includes various antibody structures, including but not limited to. Natural antibodies are molecules with various structures. For example, a native IgG antibody is here a tetrameric glycoprotein of about 150,000 Daltons, consisting of two identical light chains and two identical heavy chains that are disulfide-bonded.
  • each heavy chain comprises a variable domain (VH), also called a variable heavy domain or heavy chain variable domain, followed by three or four constant domains (CHI, CH2, CH3 and optionally CH4).
  • VH variable domain
  • VL variable domain
  • CL constant light chain
  • the light chain of an antibody can be assigned to one of two types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequence of its constant domain.
  • Antibodies in the present invention may be, but are not limited to, human antibodies, chimeric antibodies and / or humanized antibodies.
  • the chimeric antibody refers to an antibody consisting of a variable region of murine immunoglobulin and a conserved region of human immunoglobulin. Such modifications simply consist of replacing the conserved region of the human antibody with a murine conserved region, thereby producing a human / murine chimera that can have an extremely low immunogenicity to be acceptable for pharmaceutical use.
  • the humanized antibody refers to an antibody composed (partially or entirely) of amino acid sequences derived from human antibody germ cells by modifying the sequence of the antibody having a non-human complementarity determining region (CDR). Humanization of variable regions and CDRs of antibodies is accomplished by techniques well known in the art.
  • Such antibodies have human conserved regions that are required for Fc dependent effector function but are likely to elicit much less immune responses to antibodies.
  • the framework regions of the variable regions are replaced by corresponding human framework regions that have left non-human CDRs substantially intact or even replaced CDRs with sequences derived from the human genome (eg, patent application US). 2006/25885).
  • Fully ly human antibodies should be directed to the human immune system Inverse systems are produced in modified genetically modified mice.
  • the humanized antibody also comprises one or more CDRs of a human framework, a non-human antibody, wherein any conserved region present is substantially identical to the human immunoglobulin conserved region, ie at least about 85% or 90% identical, preferably Denotes an antibody wherein at least 95% are identical.
  • Antibody fragments in the present invention represent antibody fragments capable of reacting with the same antigen as the antibody counterpart.
  • Such fragments can be simply identified by one skilled in the art, including, for example, Fab fragments (eg, fragments by papain digestion), Fab 'fragments (eg, fragments by pepsin digestion and partial reduction), F (ab ') 2 fragments (eg fragments by pepsin digestion), Facb (eg fragments by plasmin digestion), Fd (eg fragments by pepsin digestion, partial reduction and recirculation), and scFv (short chain Fv' For example, fragments by molecular biology techniques).
  • Such fragments may be produced by enzyme cleavage, synthetic or recombinant techniques, as known in the art and / or disclosed herein.
  • the immunocytokine conjugated with the human interferon-beta variant according to the present invention was shown to exhibit interferon-beta activity by inducing cytotoxicity and phosphorylation of pSTAT-1, which is not seen in the antibody itself (see Examples 3 and 4).
  • the present invention also provides an immunocytokine, characterized in that the antibody or fragment thereof is an antibody or fragment thereof against an antigen selected from the group comprising a tumorous antigen and multiple sclerosis specific antigen. Tumors that grow over a certain size will need to form new blood vessels to grow further or metastasize to other sites.
  • interferon-beta has been reported to inhibit tumor cell growth by inhibiting tumor cell angiogenesis.
  • interferon-beta may induce innate or acquired immune reflexes in the surrounding environment of the site where the tumor is present, thereby inducing tumor cell death and exerting an anticancer effect. Therefore, the human interferon ⁇ beta variant according to the present invention has improved activity and function compared to the native type interferon-beta, and thus forms a cancer in the form of an immunocytokine bound to an antibody that specifically recognizes a tumor antigen.
  • the native interferon-beta may have a better therapeutic effect compared to the conjugated immunocytokine.
  • the tumorous antigen is a protein produced by tumor cells that elicit immune responsiveness, especially T-cell mediated immune responses.
  • Tumor antigens are well known in the art and include, for example, glycoma-associated antigens, cancer embryonic antigens (CEA), ⁇ -human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-banner AFP , Tyroglobulin, RAGE-1, lia-CA IX, human telomerase reverse transcriptase, RU1, RU2CAS), intestinal carboxy esterase, mut hsp70-2, M-CSF, prostatase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate-carcinoma tumor antigen -l (PCTA-l ), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF) -I, IGF-I I, IGF-I receptor and mesothe
  • the type of neoplastic antigen referred to in the present invention may also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TM).
  • TSA is specific for tumor cells and does not occur on other cells of the body.
  • TAA related antigens are expressed on normal cells under conditions that are not native to tumor cells and instead also do not elicit an immunogenic resistance state to the antigen. Expression of antigens on tumors can occur under conditions that cause the immune system to respond to antigens.
  • TAA may be an antigen expressed on normal cells during fetal development if the immune system is immature and unable to react or may be an antigen expressed normally at very low levels on normal cells but at much higher levels on tumor cells.
  • TSA or TAA antigens include: MART-1 / MelanA (MART-I), gplOO (Prael 17), tyrosinase, TRP-1, TRP-2, differentiation antigens and MAGE-1 Tumor-specific multiline antigens such as, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; Overexpressed embryonic antigens such as CEA; Overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2 / neu; Native tumor antigen resulting from chromosomal translocation; For example BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; And viral antigens such as Epstein Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7.
  • MART-1 / MelanA MART-I
  • gplOO Prael 17
  • Antibodies that specifically recognize the tumorous antigen include, for example, HuM195 (see, eg, Kossman et al., (1999) Clin. Cancer Res. 5: 2748-2755), CMA-676 (eg, Sievers et al., (1999) Blood 93: 3678-3684), AT13 / 5 (see, for example, Ellis et al. (1995) J. I : unol. 155: 925-937), HB7, trastuzumab ( For example, HERCEPTIN; Fornier et al., (1999) Oncology (Huntingt) 13: 647-58, TAB-250 (Rosenblum et al., (1999) Clin. Cancer Res.
  • HuM195 see, eg, Kossman et al., (1999) Clin. Cancer Res. 5: 2748-2755
  • CMA-676 eg, Sievers et al., (1999) Blood 93: 3678-36
  • CC49 e.g. , Pavlinkova et al. (1999) Clin. Cancer Res. 5: 2613-2619
  • B72.3 see, eg, Divgi et al., (1994) Nucl. Med. Biol. 21: 9-15
  • mouse monocle Ronald anti-HM1.24 IgG2a / ⁇ mouse monocle Ronald anti-HM1.24 IgG2a / ⁇
  • humanized anti-HM1.24 IgGl / ⁇ antibody see, eg, Ono et al. (1999) Mol. Iol uno.
  • trastuz Mab see, eg, HERCEPTIN, Fornier et al., (1999) Oncology (Huntingt) 13: 647-658
  • TAB-250 Rosenblum et al., (1999) Clin. Cancer Res. 5: 865-874
  • BACH- 250 Id.
  • TA1 see, eg, Maier et al. (1991) Cancer Res.
  • rituximab ibri Momab Tiuxetane (Ibritumotnabtiuxetan), and tosi tumomab, AME-133v (Applied Molecular Evolution), Ocrelizumab (Roche), OfaUimumab (Genmab), TRU- 015 (Trubion) and IMMU-106 (Immunomedics) Included, but not limited to.
  • the present invention need not limit the use of the antibodies described above, and other such antibodies as known to those skilled in the art can be used in the compositions and methods described herein.
  • IFN- ⁇ was first introduced as a therapeutic agent for multiple sclerosis in order to obtain an antiviral effect, but many studies have revealed its mechanism of action.
  • IFN- ⁇ inhibits antigen expression by inhibiting the activation of HLA class II molecules induced by IFN- ⁇ and prevents the activation of T cells.
  • IFN- ⁇ also inactivates Co-st imul atory molecules to inhibit T cell activation (28, 29) and induces autoreactive T cells to apoptosi s.
  • the effect of IFN- ⁇ on the brain-blood barrier is thought to inhibit T cells from adhering to vascular endothelial cells and to reduce the ability to enter the brain.
  • the human interferon-beta variant according to the present invention has improved activity and function compared to the native interferon-beta, and thus targets the form of an immunocytokine bound to an antibody that recognizes multiple sclerosis specific antigens. If used for 3 ⁇ 4, it may be better than the therapeutic effect of interferon-beta alone.
  • Multiple sclerosis specific antigens and antibodies include, for example, CD20 and the anti-chain ri tuximab that recognizes it, CD52 and the alpha receptors of the monoclonal antibodies alemtuzumab and interleukin-2 that recognize it, and dacl i horrab that recognizes it. It is not limited to this.
  • the present invention also provides an immunocytokine wherein the human interferon-beta variant is bound to the antibody or fragment thereof by a peptide linker.
  • Peptide linkers are molecules in which amino acids or amino acid-like substances connect two or more separate substances to each other by short fragments of amino acids or amino acid analogs linked to each other by peptide bonds. Glycine, serine, alanine, etc.
  • Glycine-serine linker may be used as the constituent amino acid.
  • the linker is composed of any one of SEQ ID NO: 5 to SEQ ID NO: 11 Or may include it.
  • the immunocytokines of the invention may preferably comprise a flexible linker sequence inserted between the human interferon-beta variant and the polypeptide of the antibody or fragment thereof.
  • the linker sequence allows for effective positioning of the human interferon-beta variant of the antibody or fragment thereof to allow for functional activity of both domains.
  • the linker means a peptide linker of natural origin or a peptide linker of synthetic origin.
  • the peptide linker consists of a linear amino acid chain, wherein the 20 naturally occurring amino acids are monomer building blocks.
  • the linker may have a repeating amino acid sequence or may have a sequence of naturally occurring polypeptides, such as a polypeptide having a hinge function. All peptide linkers can be encoded recombinantly because they can be encoded by nucleic acid molecules. Since the linker is itself a peptide, human interferon-beta variants and antibodies or fragments thereof are linked to the linker via peptide bonds.
  • the linker consists of amino acids linked together by peptide bonds, preferably 1-20 amino acids linked by peptide bonds, wherein the amino acids are selected from 20 natural amino acids.
  • Suitable linkers include, for example, cleavable linkers and noncleavable linkers. Cleavable linkers are typically easily cleaved under intracellular conditions. Suitable cleavable linkers include, for example, peptide linkers cleavable by intracellular proteases such as lysosomal proteases or endosomal proteases. The linker is linked to, for example, the N-terminus of the linker to the heavy chain C-terminus of the antibody.
  • Linkage of the antibody to the heavy chain C-terminus preferably results in expression of the antibody of the invention.
  • the nucleotide sequence encoding the linker sequence in the vector may be directly linked to the antibody expressed by the expression vector by concatenating the protein expression frame.
  • the linker may be linked to the light chain C-terminus of the antibody or to both the light chain C-terminus and the heavy chain C-terminus of the antibody.
  • the C-terminus of the linker is linked to the N-terminus of the interfertain beta variant of the invention.
  • the peptide linker of the present invention may be a peptide linker known in the art, but preferably may be a glycine-serine linker or a peptide linker comprising the amino acid sequence of SEQ ID NO: 5 to SEQ ID NO: 11.
  • a gly-ser linker for example, (Gly x Ser y ) z type (x is an integer of 1 to 5, y is an integer of 1 to 2, z is an integer of 1 to 6), eg For example, (gly 4 se ri ) 3 or (gly 3 ser 2 ) 3 It may be, and more preferably may be a linker represented by the amino acid sequence of GGGGS or GGGGSGGGGSGGGSG, but is not limited thereto.
  • the present invention also relates to an amino acid sequence of the human interferon-beta variant polypeptide, wherein the amino acid sequence of the antibody or fragment thereof is in the heavy chain C—terminal, light chain C-terminal or heavy and light chain C-terminal positions.
  • the amino acid sequence of the human interferon-beta variant may be at the heavy chain C-terminus, light chain C-terminus or heavy and light chain C-terminus positions relative to the amino acid sequence of the antibody or fragment thereof, preferably the amino acid of the antibody or fragment thereof At the C-terminal position relative to the sequence.
  • the present invention also provides an immunocytokine, characterized in that the immunocytokine comprises an amino acid sequence of one of the group consisting of SEQ ID NO: 12, 13, 15 or 17.
  • the present invention (a) human represented by any one of SEQ ID NO: 2 to SEQ ID NO: 4 Interferon-beta variants; (b) a peptide linker represented by any one of SEQ ID NO: 5 to SEQ ID NO: 11; And (c) an immunocytokine comprising the antibody or fragment thereof.
  • the present invention also provides a polynucleotide encoding the immunocytokine.
  • the polynucleotide may be used without limitation as long as it encodes a peptide of the immunocytokine to which the human interferon-beta variant of the present invention and an antibody or fragment thereof are bound, and include all DNA, cDNA, and RNA sequences.
  • the polynucleotide refers to a substance having a amino acid sequence represented by SEQ ID NO: 3 or encoding a peptide having an amino acid sequence having at least 70% homology with the amino acid sequence. It can be isolated from nature or prepared by known genetic engineering methods in the art.
  • the invention also provides a vector comprising said polynucleotide.
  • the vector refers to an expression vector prepared by a person skilled in the art to express the immunocytokine of the present invention by inserting the polynucleotide of the present invention into the vector according to any method known in the art and using an appropriate transcription / translational regulatory sequence.
  • the polynucleotide sequence cloned according to the present invention may be operably linked to an appropriate expression control sequence, wherein the operably linked gene sequence and expression control sequence include a selection marker and a replication icat ion origin. It can be included in one expression vector.
  • 'Operably l inked' means that the polynucleotide sequence is linked in a manner that allows gene expression to an expression control sequence.
  • the expression control sequence refers to a DNA sequence that controls the expression of a polynucleotide sequence operably linked in a particular host cell.
  • Such regulatory sequences are selected from the group consisting of a promoter for conducting transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA liposome binding site, and a sequence controlling termination of transcription and translation It can include the above.
  • the vector used as the mother vector of the expression vector is not particularly limited, and any plasmid, virus or other medium commonly used for expression in a microorganism used as a host cell in the art to which the present invention belongs can be used. .
  • the plasmids include Escherichia coli-derived plasmids (pBR322, pBR325, pUC118 and PUC119, pET-22b (+)), Bacillus subtilis-derived plasmids (pUBllO and pTP5), and yeast-derived plasmids ( ⁇ 13, ⁇ 24 and YCp50).
  • the virus may be an animal virus such as a retrovirus, adenovirus or vaccinia virus, or a nasal virus such as baclovirus, but is not limited to 3 ⁇ 4.
  • the present invention also provides a host cell transformed with the vector. The host cell may choose to modulate the expression of the inserted sequence or to advance the gene product in a particular desired manner.
  • Different host cells have characteristic and specific mechanisms for the translation and post-translational processing and modification of proteins. Suitable cell lines or host systems can be chosen to provide the desired modification and processing of the expressed heterologous protein. Expression in yeast can produce biologically active products. Expression in eukaryotic cells can increase the likelihood of "natural" folding.
  • Host cells capable of stably and continuously cloning and expressing the vector of the present invention may use any host cell known in the art, for example, E. coli JM109, E. coli BL21DE, E. coli DH5, E coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, and the like, and also Agrobacterium sp.
  • strains such as Agrobacterium A4, Bacillus subtilis Other enterobacteria such as bacilli, Salmonel la typhi murium or Serratia marcescens and various Pseudomonas genus strains such as is may be used as host cells.
  • the host cell may be a yeast (Saccharomycecerevisiae), a stromal cell and a human cell (eg, a CH0 cell line). (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and the like.
  • the host cell may be preferably a CH0 cell line.
  • the method of transforming the host cell by transferring the vector into the host cell may be any known method and is not particularly limited. For example, calcium phosphate precipitation, DEAE— dextran method, electroporation, direct microinjection, DNA—DNA loaded liposome method, lipofectamine—DNA Liofectamine-DNA complex, cell sonicat ion, gene bombardment using high velocity microprojectile, polycation. And receptor-mediated transfection. Some of these techniques can be refined for in vivo or ex vivo use.
  • the present invention also includes the steps of (a) providing a host cell, (b) culturing a provided cell, and (c) recovering an immunocytokine from said cell or culture medium to produce an immunocytokine. It provides a method for producing an immunocytokine. Cultivation of the transforming microorganism is carried out under appropriate conditions that allow for the expression of immunocytokines in which the target protein, human interferon-beta variant, and the antibody or fragment thereof is bound, is carried out according to methods well known to those skilled in the art. can do.
  • the transforming microorganism can be cultured in large quantities by conventional culture methods.
  • As the culture medium a medium consisting of a carbon source, a nitrogen source, vitamins, and minerals may be used.
  • LB medium Lia-Bertani Broth
  • Cultivation of microorganisms is possible under conventional microbial culture conditions and may be, for example, incubated for 10 to 40 hours in a temperature range of 15 ° C. to 45 ° C. Centrifugation or filtration may be performed to remove the culture medium in the culture and recover only the concentrated cells, which steps may be performed by those skilled in the art as needed.
  • the concentrated cells can be frozen or lyophilised according to conventional methods so as not to lose their activity.
  • Proteins expressed in the transforming microorganism can be purified in a conventional manner, using, for example, salting out (eg ammonium sulfate precipitation, sodium phosphate precipitation), solvent precipitation (acetone, ethanol, etc.). Protein fraction precipitation), dialysis, gel filtration, ion exchange, column chromatography such as reverse phase column chromatography, and ultrafiltration may be applied alone or in combination to purify the immunocytokines of the present invention.
  • salting out eg ammonium sulfate precipitation, sodium phosphate precipitation
  • solvent precipitation acetone, ethanol, etc.
  • Protein fraction precipitation dialysis
  • gel filtration ion exchange
  • column chromatography such as reverse phase column chromatography
  • ultrafiltration may be applied alone or in combination to purify the immunocytokines of the present invention.
  • immunocytokines conjugated with human interferon-beta variants of the present invention immunocytokines can be prepared with a significantly better efficiency than human interferon-beta conjugated immunocytokines (see Example 2). Meanwhile, the present invention is '
  • the human interferon-beta variant provides a method for increasing the production of target specific human interferon-beta, characterized in that consisting of a peptide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4.
  • target specific human interferon-beta may represent the immune cytokine of the present invention.
  • the immunocytokines comprising the human interferon-beta variants of the present invention and antibodies or fragments thereof exhibit the activity of interferon-beta and the properties of the antibodies in Hung Shi, which can be used for targeting ' treatment of multiple sclerosis or cancer, and according to the present invention.
  • Immune cytokines can be produced with superior efficiency compared to immunocytokines conjugated with native interferon-beta.
  • FIG. 1 is a result of confirming the expression level of the immunocytokine prepared in the host cell according to the present invention through Western-blot (1: culture medium, 2: B12 heavy chain-natural interferon, 3: B12 heavy chain-interferon variant) , 4: B12 light chain-natural interferon, 5: B12 light chain-interferon variant).
  • Figure 2 is a schematic diagram showing an immunocytokine conjugated human interferon-beta variants of the present invention.
  • Figure 3 shows the process of constructing pRBLX2-INF by inserting the gene sequence consisting of heavy chain -l inker-interferon in the pRBLX2 vector (left) and the gene sequence consisting of heavy chain -1 inker-interferon-beta variant in the pRBLX2 vector.
  • This is a schematic diagram showing the process (right) to insert pRBLX2-INF.
  • Figure 4 confirms the expression of the immunocytokine conjugated to the human interferon-beta variant of the present invention (right) and the immunocytokine conjugated to the human interferon-beta (left) by SDS-PAGE. At this time, each heavy and light chain is marked with ⁇ (lane 1 is a marker).
  • Figure 5 shows the protein expression of the immunocytokines conjugated with the human interferon-beta variant (lane 2) of the present invention and the immunocytokines conjugated with the control human interferon-beta (lane 1), respectively. Left) and anti-interferon antibodies (right) were identified via Western blot ol.
  • Figure 6 is the result of confirming the expression level of the immunocytokine prepared in the host cell by BCA assay (ACC # 1: B12 heavy chain-natural interferon, ACC # 2: B12 heavy chain-interferon stool Variant, ACC # 6: B12 light chain-natural interferon, ACC # 7: B12 light chain-interferon variant).
  • Figure 7 is the result of confirming whether the interferon activity of the immuno-satokine in which the human interferon-beta variant and the B12 antibody in accordance with the present invention is phosphorylated STAT-1.
  • 8 is a result of confirming the interferon-beta activity through cytotoxicity after treatment with cells for 24 hours to the immune cytokine conjugated to human interferon-beta variant and B12 antibody according to the present invention (Carbi feron: human interferon-beta Variant, B12: B12 antibody, ACC # 2: human interferon-beta variant and B12-coupled immunocytokine).
  • FIG. 9 is a result of confirming the interferon-beta activity through cytotoxicity after treating the cells with immune cytokines in which the human interferon-beta variant and the B12 antibody are combined for 48 hours according to the present invention
  • Carbi feron human interferon-beta Variant
  • B12 B12 antibody
  • ACC # 2 human interferon-beta variant and B12-coupled immunocytokine
  • Figure 10 shows a schematic diagram of an immunocytokine prepared by binding a human interferon-beta variant after binding the Rigid Hel ical linker to the ERBB2 antibody (A) and c-MET antibody (B).
  • CHO-S cells were transfected at least 5 passages at a density of 3X10 5 cells / ml to prepare for transformation. After passage, cell survival was maintained at 90% or more, and the cells were seeded at a density of 5 ⁇ 10 5 cells / ml to prepare for transformation. After 24 hours of cell seeding, the survival rate (> 9) and the cell density (1X106 eel ls / ml) were checked, and 50ug DNA was transformed into CHO-S cells incubated in 30ml culture medium using a transformation solvent. .
  • reagent A including sodium carbonate, Bicinchoninic acid, etc.
  • reagent B including 4% cupric sulfate
  • FIG. 5 shows a schematic diagram of the structure of the immunocytokine conjugated with human interferon-beta variants.
  • the linker represented by SEQ ID NO: 5 and the interferon-beta and interferon-beta variants were cloned into the heavy chain region of the antibody. Then, the restriction gene Avrl l (CCTAGG) cleavage site and the Bstzl7I (GTATAC) cleavage site were inserted at the 3'-end and 5'-end of the entire gene, respectively, to secure the final gene of the heavy chain. In addition, the final gene of the light chain was secured by inserting restriction enzyme EcoRV (GATATC) cleavage site and PacI (TTMTTAA) at the 3 '-and 5'-terminal ends of the light chain of the antibody. The process shown in Figure 3 is shown in a schematic diagram.
  • the prepared sample was loaded on a Tricine SDS-PAGE gel with a marker and subjected to electrophoresis for 1 hour and 30 minutes at a voltage of 130v. After that, the gel was carefully separated and immersed in the Coassie blue staning solut ion and shaken for 30 minutes for dyeing. After dyeing, the gel was transferred to Destaning buffer and shaken for 30 minutes to destain. Destaining was performed three times.
  • Interferon ⁇ antibody was prepared by diluting 1: 1000 in TBS-T, and anti-human IgG-HRP antibody was prepared by diluting 1: 3000 in TBS-T.
  • the membrane was immersed in an antibody dilution and reacted by shaking for 2 hours at room temperature. After this process, the mixture was wiped with TBS-T three times for 10 minutes, and reacted for 1 hour by adding a secondary antibody conjugated with horseradi sh peroxidase (HRP) at room temperature. After performing the washing process once again, the band was confirmed by ECL reagent (enhanced chemi luminescence reagent, Intron). The intensity of the band was measured using C-DiG (LI-C0R, USA).
  • lane 1 is an immunocytokine conjugated with human interferon-beta
  • lane 2 is an immunocytokine conjugated to the human interferon-beta variant.
  • Tr i c ine-SDS PAGE and Western blotting it was confirmed that the expression level of immunocytokine conjugated with human interferon-beta mutant was higher than that of human immuno-conjugated immunocytokine.
  • the culture was measured in Cedex bio (Roche, USA), respectively, for accurate comparison of expression levels.
  • the expression level of human interferon-beta conjugated immunocytokine was confirmed to be low at the concentration below the measurement range (10 mg / L or less), and the immunocytokine conjugated with human interferon-beta variant was about 32 mg / L. It was confirmed that the expression increased by at least three times.
  • 3xl0 5 0VCAR-3 cells were dispensed into each wel l of 6-wel l plate and incubated in 37.5 ° C., 5% C02 environment for 24 hours. After 24 hours, the cell cultures were removed and cultured to a concentration of 600 ng / ml of human interferon-beta mutant (600 ng / ml of human interferon-beta mutant and immunocytokine (ACC) conjugated with B12 antibody) or 1800 ng / ml. The plate was collected and treated for 1 hour, after which the plates were collected and washed three times with each wel of PBS, followed by RIPA buffer containing a protease inhibitor and a phosphatase inhibitor.
  • ⁇ was treated and placed on Ice for 30 minutes to lyse the cells.
  • the lysed cells were placed in a 1.5 ml tube and centrifuged at 13000 rpm and 4 ° C to collect only the supernatant (lysate) and collected in a new tube.
  • the protein concentration of the lysate was quantified using BCA quantification method, and 30yg lysate was collected, mixed with 5x sample buffer, and boiled at 10 (C for 10 minutes to induce protein denaturation.
  • the 3M paper was covered again, soaked in transfer buffer, and protein transfer was performed for 90 minutes at 100 volts. Blocked for 1 hour and 30 minutes in Tris-buffered saline-Tween 20 (TBS-T, 0.1% Tween20) containing 3 ⁇ 4 of BSA, and 1: 1000 in TBS-T for anti-p-STATl antibody
  • TBS-T Tris-buffered saline-Tween 20
  • the anti-GAPDH antibody was prepared by diluting 1: 3000 in TBS-T.
  • the membrane was immersed in antibody dilution and shaken at room temperature for 2 hours. After this procedure, the mixture was wiped with TBS-T three times for 10 minutes, and reacted for 1 hour by adding a secondary antibody bound to horseradish peroxidase (HRP) at room temperature. After washing again, the pu and band were treated with an ECL reagent (enhanced chemi luminescence reagent, Intron) and then developed on the film.
  • the cell cultures were removed and treated with human interferon-beta mutant (carbiferon), B12 antibody and immunocytokine at a concentration of 10-L0000ng / ml, followed by incubation for 24 hours or 48 hours. It was. After incubation for 24 hours or 48 hours, remove the culture medium, proceed with PBS wash twice, and mix WST reagent 1: 10 into the culture solution and treat ⁇ ⁇ ⁇ for each wel, followed by 37.5 ° C, After standing in a 5% C02 environment, absorbance at 430 nm was measured.
  • cytotoxicity was not found in the cell group treated with B12 antibody alone, but cytotoxicity was shown to be concentration dependent in the cell group treated with human interferon-beta and immunocytokines. It was also confirmed that it still exhibits interferon activity (FIGS. 8 and 9).
  • an immunocytokine conjugated with an interferon-beta variant was prepared as follows.
  • a Rigid Hel ical linker was coupled to a heavy chain region of an ERBB2 (Hercept in) antibody and a c-MET antibody.
  • An expression cassette was then prepared that binds human interferon-beta variants to express anti-c-Met immunocytokines (A) and anti-ERBB2 immunocytokines (B).
  • Clones were then cloned into pRBLX2, and each vector was trans-patterned into CH0-S cells, followed by incubation for 7 days. Recovery of transfect ions, cultures and expressions was performed as described in Example 4.
  • the expression levels of CH0-S cells were compared, the expression levels of the immunocytokines conjugated with the human interferon-beta conjugated to the c— Met antibody or the ERBB2 antibody and the immunocytokines conjugated with the human interferon-beta variant were compared.
  • the expression levels of immunocytokines conjugated with human interferon-beta variants were higher and that interferon activity was better.
  • the expression of the human interferon-beta variant of the present invention is very well compared to the wild type interferon beta.
  • immune cytokine beta variants are bonded-human interferon in accordance with the present invention. It can be used as a target therapeutic agent for diseases such as multiple sclerosis and cancer in that both the interferon activity superior to the native interferon-beta and the characteristics of the antibody that recognize a specific antigen are shown. Compared to the immunocytokine, the industrial efficiency is excellent because the production efficiency is significantly higher.

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Abstract

La présente invention concerne une immunocytokine dans laquelle un variant d'interféron bêta humain est conjugué à un anticorps ou à un fragment de ce dernier, et un procédé de préparation de cette dernière. Le variant d'interféron bêta humain présente une activité ou des fonctions supérieures par rapport à l'interféron bêta naturel, et la productivité de l'immunocytokine selon la présente invention est excellente. En outre, l'immunocytokine selon la présente invention peut être avantageusement utilisée en tant qu'agent thérapeutique cible pour la sclérose en plaques ou le cancer étant donné que l'immunocytokine exprime à la fois les fonctions de l'interféron bêta et les caractéristiques de l'anticorps se liant à un antigène spécifique.
PCT/KR2016/002129 2015-03-03 2016-03-03 Immunocytokine conjuguée à un variant d'interféron bêta humain et son procédé de préparation WO2016140528A1 (fr)

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JP2017546891A JP6779896B2 (ja) 2015-03-03 2016-03-03 ヒトインターフェロン−β変異体が接合された免疫サイトカインとその製造方法
ES16759164T ES2839404T3 (es) 2015-03-03 2016-03-03 Inmunocitocina conjugada con variante de interferón-beta humana y procedimiento de preparación de la misma
CN201680015754.1A CN107438624B (zh) 2015-03-03 2016-03-03 人干扰素-β变体缀合免疫细胞因子及其制备方法
EP16759164.3A EP3266801B1 (fr) 2015-03-03 2016-03-03 Immunocytokine conjuguée à un variant d'interféron bêta humain et son procédé de préparation
US15/693,148 US10806799B2 (en) 2015-03-03 2017-08-31 Human interferon-beta variant conjugated immunocytokine and method for preparing same
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KR102371151B1 (ko) * 2020-03-13 2022-03-07 주식회사 큐로셀 항-bcma 결합 영역, 이를 포함하는 융합단백질, 및 이를 포함하는 조성물
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